Use of bismuth subgallate in inhibition of production of nitric oxide synthase

ABSTRACT

The present invention discloses the new use of bismuth subgallate for use in the inhibition of the production of nitric oxide synthase. Also disclosed is the synergistic efficacy of bismuth subgallate in combination with borneol in the inhibition of the production of nitric oxide synthase.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a new use of bismuth subgallatein the inhibition of the production of nitric oxide synthase.

[0003] 2. Description of the Prior Art

[0004] Nitric oxide (NO) is an unstable free radical that mediates bothhomeostatic and pathophysiologic processes within the cardiopulmonary,nervous, and immune systems. The list of potential disease associationsfor NO is increasing dramatically (Cochran et al., Medicinal ResearchReviews, 1996, 16(6):547-563). Thus, agents that modulate the activityof NO may be of considerable therapeutic value. In particular, thosethat reduce the formation of NO may be beneficial in pathophysiologicalstates in which excessive production of NO is a contributory factor.These include diseases such as septic shock, neurodegenerativedisorders, and inflammation. NO is formed endogenously by a family ofenzymes known as nitric oxide synthases (NOS) (Hobbs et al., Annu. Rev.Pharmacol. Toxicol, 1999, 39:191-220).

[0005] It is known that three distinct isoforms of NOS have beenidentified: an inducible form (iNOS) and two constitutive forms referredto neuronal NOS (nNOS) and endothelial NOS (eNOS). The NOS, particularlyiNOS, are associated with the conditions including platelet aggregation,homeostatic processes, tissue injury, inflammatory conditions, shockstates, immune disorders, disorders of gastrointestinal motility anddiseases of the central and peripheral nervous system and diabetes(Epstein, The New England Journal of Medicine, 1993, 329(27): 2002-2011;Hobbs. et al, Annu. Rev. Pharmacol. Toxicol., 1999, 39: 191-220;Pfeilschfter et al, Cell Biology International, 1996, 20(1): 51-58). Itis believed that neuropathy is closely related to diabetic ulcers andthus the neuropathy is a major contributing factor to most lowerextremity diabetic ulcers (N. Engl J Med, Volume 343 (11), Sep. 14,2000, pp. 787-793; and J. Fam. Pract., Volume 49(11) Supplement,November 2000, pp. S40-S48). The NOSs are also associated with theconditions caused by nitric oxide, such as tissue injury, cerebralischemia, epilepsy, immunity and inflammation (Mulligan et al., Proc.Natl. Acad. Sci. USA, 1991, 88:6338-6342).

[0006] Given the above, it is expected that the compounds or agentscapable of decreasing the amount or activity of NOS are useful astherapeutic agents. For example, U.S. Pat. No. 6,030,985 discloses theamidine derivatives useful for treating and preventing conditions inwhich inhibition of nitric oxide synthase is beneficial, such as stroke,schizophrenia, anxiety, and pain. U.S. Pat. No. 6,071,906 provides apharmaceutical compositions containing an amidino derivative useful asan inhibitor of nitric oxide synthase. U.S. Pat. No. 6,133,306 featurestreating a neurodegenerative disease by administration of an effectiveamount of a nitroindazole, which is an inhibitor of neuronal nitricoxide synthase. U.S. Pat. No. 6,235,747 relates to certain6-phenyl-pyridin-2-ylamine derivatives that exhibit activity as nitricoxide synthase (NOS) inhibitors, to pharmaceutical compositionscontaining them and to their use in the treatment and prevention ofcentral nervous system disorders.

[0007] Bismuth subgallate is the product of the reaction among gallicacid, glacial acetic acid and bismuth nitrate which is represented by achemical formula of C₇H₅BiO₆. It is known as an oral anti-diarrhea agenteffective in treating acute or chronic diarrhea by virtue that it canreact with H₂S, which is present in large quantities in the intestinaltract due to abnormal fermentation, and thereby alleviate diarrhea andpains caused by gas irritation to the intestinal tract. Bismuthsubgallate can also be used as a disinfectant in view of its nature as abenzene derivative.

[0008] A pharmaceutical composition for wound healing comprising bismuthsubgallate and borneol is disclosed in U.S. Pat. No. 6,232,341. However,none of the prior art teaches or suggests the new use of bismuthsubgallate in the inhibition of NO synthase.

SUMMARY OF THE INVENTION

[0009] It is surprisingly found that bismuth subgallate is useful in theinhibition of the production of nitric oxide synthase (NOS).

[0010] Accordingly, the invention provides the new use of bismuthsubgallate in the inhibition of the production of NOS.

[0011] An object of the invention is to provide a method of inhibitingthe production of nitric oxide synthases (NOS) in a subject, comprisingadministering to said subject in need thereof an effective amount ofbismuth subgallate; wherein said bismuth subgallate is administered inan amount effective to inhibit the production of NOS. According to thepreferred embodiment of the invention, the method comprisesadministering to said subject in need thereof an effective amount ofbismuth subgallate in combination with borneol, wherein said bismuthsubgallate in combination with borneol are administered in an amountsynergistically effective to inhibit the production of NOS.

[0012] Another objective of the invention is to provide a pharmaceuticalcomposition for inhibiting the production of nitric oxide synthasescomprising an effective amount of bismuth subgallate sufficient toinhibit the production of NOS and a pharmaceutical acceptable carrier.According to a preferred embodiment of the invention, the pharmaceuticalcomposition of the invention further comprises borneol to provide asynergistic effect in the inhibition of the production of NOS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows the effect of bismuth subgallate on NO production bythe activated RAW 264.7 cells.

[0014]FIG. 2 shows the effect of borneol on NO production by theactivated RAW 264.7 cells.

[0015]FIG. 3 shows the effect of bismuth subgallate in combination withborneol on NO production by the activated RAW 264.7 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to a new use of bismuth subgallatein the inhibition of the production of nitric oxide synthases. It issurprisingly found in the invention that bismuth subgallate is useful inthe inhibition of the production of nitric oxide synthase. Inparticular, the claimed invention provides a method of inhibiting theproduction of nitric oxide synthases (NOS) in a subject, comprisingadministering to said subject in need thereof an effective amount ofbismuth subgallate, wherein said bismuth subgallate is administered inan amount effective to inhibit the production of NOS. In addition, it isfound that bismuth subgallate in combination with borneol exhibit asynergistic effect in the inhibition of the production of nitric oxidesynthase. In particular, the claimed invention provides a method ofinhibiting the production of nitric oxide synthases (NOS) in a subject,comprising administering to said subject in need thereof ansynergistically effective amount of bismuth subgallate in combinationwith borneol, wherein said bismuth subgallate in combination withborneol are administered in an amount synergistically effective toinhibit the production of NOS.

[0017] Definition

[0018] The term “bismuth subgallate” as used herein, refers to theproduct of the reaction among gallic acid, glacial acetic acid andbismuth nitrate which is represented by a chemical formula of C₇H₅BiO₆.

[0019] The term “borneol” as used herein, refers to the product isolatedfrom Dryobalanops aromatica or the like and represented by the chemicalformula C₁₀H₁₇OH.

[0020] The term “effective amount” as used herein refers to an amountsufficient to provide an effect sufficient for the inhibition of theproduction of nitric oxide synthases to bring improvement in patients.

[0021] The term “carrier” as used herein refers to a diluent, anexcipient, a recipient and the like for use in preparing admixtures of apharmaceutical composition.

[0022] Pharmaceutical Composition

[0023] The invention provides a pharmaceutical composition for use inthe inhibition of the production of nitric oxide synthase, whichcomprises an effective amount of bismuth subgallate sufficient toinhibit the production of NOS and a pharmaceutical acceptable carrier.

[0024] The pharmaceutical composition preferably comprises bismuthsubgallate at an amount ranging from 0.05 to 40 percent by weight. Morepreferably, the amount of bismuth subgallate in the pharmaceuticalcomposition according to the present invention is from 1 to 20 percentby weight. Most preferably, the amount of bismuth subgallate in thetopical pharmaceutical composition according to the present invention isfrom 2 to 10 percent by weight.

[0025] According to the invention, the pharmaceutical compositionfurther comprising borneol is synergistically effective in theinhibition of nitric oxide synthases. Preferably, the pharmaceuticalcomposition comprises bismuth subgallate at an amount ranging from 1 to30 percent by weight and borneol at an amount ranging from 0.05 to 10percent by weight. More preferably, the amounts of bismuth subgallateand borneol in the pharmaceutical composition are from 3 to 15 percentby weight, and from 0.1 to 5 percent by weight, respectively. Mostpreferably, the amounts of bismuth subgallate and borneol in thepharmaceutical composition are from 4 to 8 percent by weight and from0.5 to 1 percent by weight, respectively.

[0026] Apart from the above-mentioned active ingredients, thepharmaceutical composition according to the present invention mayfurther comprise other traditional agents which are helpful in theinhibition of nitric oxide synthases, such as the compounds disclosed inWO 94/12165, WO 94/14780, WO 93/13055, EP 0446699A1, U.S. Pat. No.5,132,453, U.S. Pat. No. 6,030,985, U.S. Pat. No. 6,071,906, U.S. Pat.No. 6,133,306 and U.S. Pat. No. 6,235,747. The incorporation of thesetraditional agents into the pharmaceutical composition according to thepresent invention is readily available for ordinary persons skilled inthe art.

[0027] According to the invention, suitable doses of the pharmaceuticalcomposition according to the invention may be determined routinely bythe medical practitioner or other skilled persons, and include therespective doses discussed in the prior art disclosing bismuthsubgallate and borneol that are mentioned hereinbefore. The disclosuresare hereby incorporated by reference.

[0028] In any event, a physician, or a skilled person, will be able todetermine the actual dosage which will be most suitable for anindividual patient. The dosage is likely to vary depending on thecondition that is to be treated, as well as the age, weight, sex andresponse of the particular patient to be treated.

[0029] According to the invention, the pharmaceutical composition can beformulated for topical, oral, parenteral or other mode ofadministration. Suitable pharmaceutical carriers used in thepharmaceutical composition of the invention include, but are not limitedto, water, salt solutions, alcohols, polyethylene glycols, gelatin,carbohydrates (such as lactose, amylose or starch), magnesium stearate,talc, silicic acid, viscous paraffin, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrrolidone, etc.

[0030] Injectable, sterile solutions, preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants, includingsuppositories are particularly suitable for parenteral application.Ampoules are convenient unit dosages. Oral applications are preferablyadministered in the forms of capsules, tablets and/or liquidformulations.

[0031] For topical applications, the pharmaceutical composition of theinvention is preferably administered in the forms of paste, cream andgel paste etc. More preferably, the pharmaceutical composition mayfurther comprise anti-inflammatory agents, astringents, emollients oranalgesics.

[0032] Utility

[0033] According to the invention, the pharmaceutical composition can beused in the inhibition of the production of nitric oxide synthase.Therefore, the pharmaceutical composition is effective in the treatmentof the diseases in which nitric oxide production is implicated, such asplatelet aggregation deficiency, homeostatic process disorder, tissueinjury, migraine, inflammatory diseases (e.g., asthma), stroke, acuteand chronic pain, hypovolemic shock, traumatic shock, reperfusioninjury, Crohn's disease, ulcerative colitis, septic shock, multiplesclerosis, AIDS associated dementia, neurodegenerative diseases, neurontoxicity, Alzheimer's disease, chemical dependencies and addictions(e.g., dependencies on drugs, alcohol and nicotine), emesis, epilepsy,anxiety, psychosis, head trauma, adult respiratory distress syndrome(ARDS), morphine induced tolerance and withdrawal symptoms, inflammatorybowel disease, osteoarthritis, rheumatoid arthritis, dilatedcardiomyopathy, acute spinal cord injury, Huntington's disease,Parkinson's disease, glaucoma, macular degeneration, diabeticneuropathy, diabetic ulcers and cancer in a mammal, including a human.

[0034] More preferably, the pharmaceutical composition of the inventionis useful in treating the conditions associated with plateletaggregation, homeostatic processes and tissue injury. The treatment ofthe condition of platelet aggregation has the effect on rapidhemostasis.

[0035] The following examples further illustrate the present invention,but are not intended to limit the scope of the present invention. Themodifications and substitutions known to those skilled in the art arestill within the scope and spirit of the present invention.

EXAMPLES Example 1 Effect of Bismuth Subgallate on Production of NitricOxide

[0036] The cells of the mice macrophage cell line RAW 264.7 (underaccession number CCRC 60001), which was obtained from Cell CultureResearch Center, Food Industry Research and Development Institute,Hsinchu, Taiwan, R.O.C., were used for the test.

[0037] The RAW 264.7 cells were incubated in a 96 well tissue cultureplate (Falcon). After 24 hours, the cells were activated with Dulbecco'sModified Eagle Medium (DMEM, 2014, GibcoBRL) in the presence and absenceof 100 ng/ml LPS, 100 U/ml IFN-γ and various concentrations (10 μM, 20μM, 50 μM, 100 μM and 200 μM) of bismuth subgallate (Hwang et al., 1994,J. Biol. Chem. 269, p. 711-715). After 24 hours, 50 μl of thesupernatant from each well was assayed for the presence of nitritethrough the Saville modification of the Griess reaction (Green et al.,Analytical Biochem, 1982, 126:131-138). The results were represented bythe means plus standard errors values (Mean±SE) of three repetitions.

[0038] As shown in FIG. 1, the cells, which were not activated, by LPSand IFN-γ did not produce NO. In contrast, the cells, which wereactivated, by LPS and IFN-γ produced up to 65 μM of NO. Furthermore,those from cells incubated in the presence of bismuth subgallate showeda reduction in nitrite concentration, reflecting the reduction in NOproduction. The production of NO was inversely proportional to theincrease in the concentration of bismuth subgallate.

Example 2 Effect of Borneol on Production of NO

[0039] The effect of borneol on the inhibition of NO synthase was testedthrough the same experimental procedures as described in Example 1.Various concentrations of borneol (0 μM, 40 μM, 80 μM, 160 μM, 320 μMand 640 μM) were used to replace bismuth subgallate of Example 1. Theresults were represented by the mean values of three repetitions withstandard errors (Mean±SE). As shown in FIG. 2, the production of NO wasnot significantly inhibited by the borneol.

Example 3 Synergistic Effect of Bismuth Subgallate in Combination withBorneol on Production of NO

[0040] The effect of bismuth subgallate in combination with borneol onthe inhibition of NO synthase was tested through the similarexperimental procedures as described in Example 1. The followingconcentrations of Bismuth subgallate: 0 μM, 12.5 μM, 25 μM, 50 μM and100 μM, were combined with the following concentrations of borneol: 0μM, 10 μM, 20 μM and 40 μM, respectively, to obtain the combinations forthe replacement of bismuth subgallate used in Example 1. The resultswere represented by the mean values of three repetitions with standarderrors (Mean±SE). As shown in FIG. 3, the production of NO wassignificantly lowered by the treatment of the combination of bismuthsubgallate with borneol. The effect of the combinations on theproduction of NO was more significant than that of bismuth subgallateonly. In other words, a combination of bismuth subgallat with borneolexhibits a synergistic effect on inhibiting NO synthase.

What is claimed is:
 1. A method of inhibiting the production of nitric oxide synthases (NOS) in a subject, comprising administering to said subject in need thereof bismuth subgallate in an amount effective to inhibit the production of NOS.
 2. The method of claim 1, further comprising administering to said subject in need thereof borneol in an amount synergistically effective to inhibit the production of NOS.
 3. A pharmaceutical composition for use in the inhibition of the production of NOS, which comprises a bismuth subgallate for use in the inhibition of the production of NOS in an effective amount sufficient to inhibit the production of NOS and a pharmaceutical acceptable carrier.
 4. The pharmaceutical composition of claim 3, wherein the amount of bismuth subgallate ranges from 0.05 to 40 percent by weight.
 5. The pharmaceutical composition of claim 4, wherein the amount of bismuth subgallate ranges from 1 to 20 percent by weight.
 6. The pharmaceutical composition of claim 5, wherein the amount of bismuth subgallate ranges from 2 to 10 percent by weight.
 7. The pharmaceutical composition of claim 3 further comprising borneol, wherein the amounts of bismuth subgallate and borneol are synergistically effective for inhibiting the production of NOS.
 8. The pharmaceutical composition of claim 7, wherein the amount of bismuth subgallate and that of borneol range from 1 to 30 percent by weight and from 0.05 to 10 percent by weight, respectively.
 9. The pharmaceutical composition of claim 8, wherein the amount of bismuth subgallate and that of borneol range from 3 to 15 percent by weight and from
 0. 1 to 5 percent by weight, respectively.
 10. The pharmaceutical composition of claim 9, wherein the amount of bismuth subgallate and that of borneol range from 4 to 8 percent by weight and from 0.5 to 1 percent by weight, respectively
 11. The pharmaceutical composition of claim 3, which further comprises another NOS inhibitor.
 12. The pharmaceutical composition of claim 7, which further comprises another NOS inhibitor.
 13. The pharmaceutical composition of claim 3, which is administered in topical, oral or parenteral route.
 14. The pharmaceutical composition of claim 7, which is administered in topical, oral or parenteral route.
 15. A method of treating diseases in a subject in which nitric oxide production is implicated, comprising administering to said subject in need thereof an effective amount of bismuth subgallate.
 16. The method of claim 15, further comprising administering to said subject in need thereof a borneol in an amount synergistically effective to inhibit the production of NOS.
 17. The method of claim 15, wherein the diseases are selected from the group consisting of: platelet aggregation deficiency, homeostatic process disorder, tissue injury, migraine, inflammatory diseases, stroke, acute and chronic pain, hypovolemic shock, traumatic shock, reperfusion injury, Crohn's disease, ulcerative colitis, septic shock, multiple sclerosis, AIDS associated dementia, neurodegenerative diseases, neuron toxicity, Alzheimer's disease, chemical dependencies and addictions, emesis, epilepsy, anxiety, psychosis, head trauma, adult respiratory distress syndrome (ARDS), morphine induced tolerance and withdrawal symptoms, inflammatory bowel disease, osteoarthritis, rheumatoid arthritis ovulation, dilated cardiomyopathy, acute spinal cord injury, Huntington's disease, Parkinson's disease, glaucoma, macular degeneration, diabetic neuropathy, diabetic ulcers and cancer in a mammal.
 18. The method of claim 15, wherein the disease is platelet aggregation deficiency.
 19. The method of claim 15, wherein the disease is homeostatic process disorder.
 20. The method of claim 18, wherein the treatment of homeostatic process disorders has an effect on rapid hemostasis.
 21. The method of claim 15, wherein the disease is tissue injury.
 22. The method of claim 15, wherein the disease is inflammatory disease.
 23. The method of claim 22, wherein the inflammatory disease is asthma.
 24. The method of claim 15, wherein the disease is a diabetic ulcer. 